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PCR amplification of Community DNA - (Jul/02/2006 )

Hi,
I am grad student working with bacterial communities from marine water. I had extracted genomic DNA by filtrating, Freeze-thaw, proteinase K and isopropanol precipitation. I am having hard time in amplifying genomic DNA with 16Sr Primers.
I have 2 questions:
1) I tried Qiaex II gel extraction kit to purify the gel product .After extraction when I ran gel I saw nothing where did I loose the product. DNA concentration was 5ug and elution was 30ul water and gel was run with 5ul of purified product??

2) Genomic DNA is contaminated with humic acids so unable to amplify with 16Sr DNA primers.
I tried diluting the sample and gel extraction& purification with no success. Any suggestions how can I get rid of humic acids
Thanks in advance
envt 007

-envt 006-

Did you start with colonies of bacteria? if so you can add a colony directly to your PCR mix and get amplification. I have done it with 16S primers myself and gotten good results.

If you don't have the colonies, you may have to clone them in order to get enough DNA. I'm not sure of the protocol, but if you search for it I'm sure you will find it.

I would suggest using an organic extraction or Qiagen makes a kit called the DNeasy tissue kit that works well and is easy.

Hope this helps.

-mel4n6-

QUOTE (mel4n6 @ Jul 3 2006, 01:58 PM)
Did you start with colonies of bacteria? if so you can add a colony directly to your PCR mix and get amplification. I have done it with 16S primers myself and gotten good results.

If you don't have the colonies, you may have to clone them in order to get enough DNA. I'm not sure of the protocol, but if you search for it I'm sure you will find it.

I would suggest using an organic extraction or Qiagen makes a kit called the DNeasy tissue kit that works well and is easy.

Hope this helps.

Hi,
I don't have colonies I have community DNA which I extracted from soil sediments by Phenol -chlorofom -isoamyalcohol and followed by isopropanol precipitation. When I run the gel I see DNA but PCR troubles me. with no amplification.
Anyway going to try Qiagen DNAse kit after Phenol- chloroform -isoamyalcohol extraction.
Thank you,
envt 007

-envt 006-

Hi,

Did you design the 16s primers? If so, there could be a problem with your primers. Have you run primer-dimer and hairpin checks on them?

_hank

-haringsh-

A common problem is adding far too much template DNA to a PCR reaction. Recall that you can amplify a single molecule. I would suggest retrying your PCR with 100x, 1000x and 10000x dilutions of your extracted DNA.
You could also determine if inhibitors were the problem by taking a known working DNA sample and adding it to your extracted DNA. If you can amplify thet sample, you know inhibition is not the issue. (I assume you have a working positive example of amplification with your 16S primers. If not, that is the first order of business).
I was confused by your first comment about using Qiaex II to extract DNA from a gel. Your genomic DNA will not go into a gel, unless it is highly degraded, so it is not surprising you saw little or nothing.

You might want to check the articles listed here:
http://openwetware.org/wiki/Bacterial_species_identification

-phage434-

Hi,
I try to amplify genomic DNA of dry rot fungi in wood, and also have inhibition problems by humic acid. I have been helped most by adding 400 ng/microliter nonacetylated BSA to my PCR reaction (Sigma, A-7906, do not use BSA for molecular biology!). Here is a helpful publication: Kreader A. (1996) Applied and Environmental Microbiology 62 (3): 1102-1106. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein.

-karen steeghs-

In our Lab we use the Mo-bio Power soil kits. The difference compared to QIagen kit was striking for soil and fecal samples.

-Canalon-